Project Summary Hematopoietic stem cell transplant (HSCT) and gene therapy are increasingly utilized for definitive management of disorders affecting the lymphohematopoietic compartment. Conditioning regimens are one of the critical components HSCT. The majority of conditioning regimens used today are either based on alkylating chemotherapy, such as busulfan, or involve varying doses of total body irradiation (TBI). These agents are genotoxic and affect organs other than the hematopoietic compartment, leading to myriad acute and long-term HSCT associated complications. Our overarching goal is to develop a non-genotoxic conditioning regimen targeting hematopoietic stem and progenitor cells (HSPCs). Preliminary data from our lab indicates MPL (thrombopoietin receptor), a critical survival signal in hematopoietic stem cells (HSCs), could be an ideal candidate for development of HSPC-targeted HSCT conditioning approaches. Restricted expression profiles within the hematopoietic compartment, with a higher expression in HSC when compared to the hematopoietic progenitor compartment, make MPL a very attractive target. We have developed two strategies to target the MPL expressing HSPC compartment. First, an immunotoxin approach, generated by conjugating MPL antibody to a ribosomal toxin, saporin (MPL-ab-SAP), and the second, a MPL receptor engaging thrombopoietin based CAR-T cell (TPO-CAR-T cells). We hypothesize that a conditioning agent targeting the MPL receptor will result in selective HSPC depletion thereby creating adequate niche space to allow donor engraftment and will have minimal risk of cytotoxicity outside the hematopoietic compartment. We will determine the mechanism, and critical determinants of MPL-ab-SAP-mediated cytotoxicity in the HSC compartment. Then, we will determine whether HSPC depletion by MPL-ab-SAP could be utilized as an effective conditioning strategy for HSCT and autologous HSC-based gene therapy. The role of TPO-CAR-T cells as an HSCT conditioning will also be studied. The K08 Award will be used to protect my time for research, help broaden my research skills and knowledge, and provide an outstanding basis for a career in basic and translational HSCT and gene therapy. This mentored phase of training will allow me to further integrate and build on my research skills as a physician-scientist. Over the next five years, I will (1) gain expertise in molecular methods needed for development of therapeutic antibodies, and gene therapy based therapeutics, (2) gain further exposure in HSC biology, transplant immunology, and murine HSCT, (3) acquire skills in cell imaging and bioinformatics, (4) acquire skills in translational research methodology, grantsmanship and leadership, and (5) build on my publication record and develop research ideas for independent investigator grants. To accomplish these goals, I have developed a well-structured training plan, and I have assembled a highly-qualified mentorship team of Dr. Spencer (expertise in cell and gene therapy), Dr. Bunting (HSC biology), Dr. Waller (HSPC biology and clinical transplantation), and Dr. Larsen (transplant immunology) at Emory University.